Manufacturing recombinant proteins at industrially relevant levels requires technologies that can engineer stable, high-expressing cell lines rapidly, reproducibly, and with relative ease. Commonly used methods incorporate transfection of mammalian cell lines with plasmid DNA containing the gene of interest. Identifying stable, high-expressing transfectants is normally laborious and time consuming. To improve this process, the ACE System has been developed based on pre-engineered artificial chromosomes with multiple recombination acceptor sites. This system allows targeted integration of single or multiple gene copies and eliminates the need for random integration into native host chromosomes. To illustrate the usefulness of the ACE System in generating stable, high-expressing cell lines, we present several case studies covering CHO cell lines expressing monoclonal antibodies.

Many methods are currently available for producing cell lines that express recombinant proteins. Most of them use plasmid transfection, or viral transduction procedures, to incorporate DNA sequences containing the gene of interest into mammalian cell lines. These processes often result in transfectants with highly variable protein expression due to random integration of the DNA into the host genome. Furthermore, these methods may necessitate time-consuming amplification events, or re-infection, to boost the cell's productivity. As a result, the process of generating and selecting a high-expressing, stable clonal cell line suitable for the clinical and commercial manufacture of biopharmaceuticals can be labor intensive and extremely time consuming.

To increase the speed and efficiency of generating high-expressing, stable cell lines for the manufacture of recombinant proteins, Chromos has developed a novel cell-line engineering system, the ACE System, based on the company's proprietary artificial chromosome technology. The system differs from conventional technologies. It facilitates the targeted and reproducible integration of multiple copies of genes into specific sites on an artificial chromosome that resides in a production cell line without amplification. These artificial chromosomes, or ACEs, contain fully functional centromeres and telomeres, and as a result are as mitotically stable as the host chromosomes. Ultimately, this results in the generation—with minimal screening and reduced timelines—of high-expressing stable clonal cell lines with high levels of gene expression. ACEs also can be purified to homogeneity by flow cytometry and readily transferred to a variety of cell types using commercially available transfection agents. This feature enables the auditioning of alternative cell lines for improved product quality or quantity, thereby providing an option not typically found in conventional mammalian cell line engineering technologies.

THE ACE SYSTEM

Core Components

Figure 1

The ACE System was designed so that one or more genes could be reliably and reproducibly loaded, with relative ease, onto an existing ACE and screened for incorporation and expression. For mammalian cell line engineering, the ACE System consists of four main components (Figure 1):

3. ACE Targeting Vector (ATV): A plasmid that contains a single recombination donor site for recombination into the acceptor sites on the Platform ACE, selection marker, and the gene(s) of interest along with all genetic elements required for enhanced expression in CHO cells.

4. ACE Integrase: A site-specific DNA recombinase that catalyzes the targeted integration of the ATV onto the Platform ACE residing in the Platform ACE Cell Line.